Hornby et al., U.S. Pat. No. 4,238,565, disclose a specific binding assay for determining a ligand (L) in a liquid medium. In several examples, the labelled conjugate is FAD, which is a prosthetic group residue (Pr), coupled to L through a linking group (R). In such an assay, a known quantity of the labelled conjugate, Pr-R-L, is added to a liquid medium, for example, a blood sample, which contains an unknown quantity of a ligand, for example, theophylline. A known titer of a ligand binding component (B), for example, theophylline antiserum, is also added. B attaches in a competitive reaction to L and Pr-R-L to form L-B and Pr-R-L-B, respectively. Then, a known amount of glucose oxidase apoenzyme (Apo) is added to the medium. The apoenzyme binds to Pr-R-L, forming a holoenzyme, Apo-Pr-R-L, which is an active enzyme. Apo is sterically inhibited, however, from binding to Pr-R-L-B due to the presence of B. Thus, the amount of holoenzyme formed is proportional to the amount of L in the medium.
The amount of holoenzyme glucose oxidase is measured by its activity in the presence of glucose, the enzyme substrate. Glucose is converted through the action of the enzyme, among other products, to hydrogen peroxide which can be measured colorimetrically by known techniques.
Carrico et al., U.S. Pat. No. 4,171,432, disclose a labelled conjugate comprising FAD as the prosthetic group residue and an iodothyronine as the ligand for use in the specific binding assay disclosed in U.S. Pat. No. 4,238,565, issued to Hornby et al.
The above-described specific binding assay is typically carried out at about 25.degree. C. using about 0.1M glucose as substrate for the glucose oxidase. This amount of glucose is about four times the known K.sub.m for glucose oxidase at 25.degree. C., K.sub.m being the substrate concentration at which the rate of enzyme activity is one-half the maximum rate, and is the amount conventionally used in enzyme-enzyme substrate reactions. The assay is accurate, easily carried out and reproducible. However, the assay does not produce quantifiable results when carried out at high temperature, e.g., about 37.degree. C. which is the temperature at which certain automated clinical analyzers operate. Therefore, an improvement which enhances the sensitivity of the assay system at 37.degree. C. is highly desirable.
Dixon et al., "Enzymes," 137-138, Academic Press (1979), state that, in some cases, an enzyme substrate can act as an enzyme activator. A well studied example of activation of an enzyme by a substrate is activation of phenylalanine hydroxylase by phenylalanine as reported as Shiman, J. Biol. Chem., Volume 255, 10029 (1980). Massey et al., J. Biol. Chem., Volume 241, 3417 (1966) postulate that binding of D-amino acid oxidase substrate to the enzyme promotes a conformational rearrangement of the enzyme which is required for full enzyme activity.